Abstract：
The ability of magnetic tweezers to apply forces and measure molecular displacements has resulted in its extensive use to study the activity of enzymes involved in various aspects of nucleic acid metabolism. These studies have led to the discovery of key aspects of protein-protein and protein-nucleic acid interaction, uncovering dynamic heterogeneities that are lost to ensemble averaging in bulk experiments. The versatility of magnetic tweezers lies in the possibility and ease of tracking multiple parallel single-molecule events to yield statistically relevant single-molecule data. Moreover, they allow tracking both fast millisecond dynamics and slow processes (spanning several hours). In this chapter, we present the protocols used to study the interaction between E. coli SSB, single-stranded DNA (ssDNA), and E. coli RecQ helicase using magnetic tweezers. In particular, we propose constant force and force modulation assays to investigate SSB binding to DNA, as well as to characterize various facets of RecQ helicase activity stimulation by SSB.
摘要：
磁性镊子施加力和测量分子位移的能力已导致其广泛用于研究涉及核酸代谢各个方面的酶的活性。这些研究发现了蛋白质-蛋白质和蛋白质-核酸相互作用的关键方面，发现在批量实验中由于集合平均而丢失的动态异质性。磁镊子的多功能性在于跟踪多个平行的单分子事件以产生统计相关的单分子数据的可能性和便利性。此外，它们允许跟踪快速的毫秒动态和缓慢的过程（跨越几个小时）。在这一章中,我们提出了用于研究大肠杆菌SSB之间相互作用的协议，单链DNA(ssDNA),和大肠杆菌RecQ解旋酶使用磁镊子。特别是,我们提出了恒力和力调制试验来研究SSB与DNA的结合，以及表征SSB刺激的RecQ解旋酶活性的各个方面。
